title: Dissection of conformational conversion events during prion amyloid fibril formation using hydrogen exchange and mass spectrometry.
abstract: A molecular understanding of prion diseases requires an understanding of the mechanism of amyloid fibril formation by the prion protein. In particular, it is necessary to define the sequence of the structural events describing the conformational conversion of monomeric PrP to aggregated PrP. In this study, the sequence of the structural events in the case of amyloid fibril formation by recombinant mouse prion protein at pH7 has been characterized by hydrogen-deuterium exchange and mass spectrometry. The observation that fibrils are substantially more stable to hydrogen-deuterium exchange than is native monomer allows both forms to be quantified during the course of the aggregation reaction. Under the aggregation conditions utilized, native monomeric protein and amyloid fibrils are the only forms of the protein detectable during the course of the fibril formation reaction, suggesting that monomer directly adds on to the fibril template. Conformational conversion is shown to occur in two steps after the binding of monomer to fibril, with helix 1 unfolding only after helices 2 and 3 transform into β-sheet. Local stability in the β-sheet core region (residues ~159-225) of the fibrils is shown to be sequence dependent in that it varies along the length of the core, and local stability in protein molecules that are ordered in the structurally heterogeneous sequence segment 109-132 is shown to be similar to that in the core. This new understanding of the structural events during prion protein aggregation has important bearing on our comprehension of the molecular basis of prion pathogenesis.
authors.affiliation: National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
authors.forename: Jogender
authors.initials: J
authors.lastname: Singh
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
mesh_terms: D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D041961:Deuterium Exchange Measurement; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D000072002:Prion Proteins; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D000682:Amyloid; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D006859:Hydrogen
keywords: AFM; C-terminal domain; CTD; GdnHCl; HDX; MS; N-terminal domain; NTD; ThT; amyloid fibrils; atomic force microscopy; guanidine hydrochloride; hydrogen exchange; hydrogen–deuterium exchange; mass spectrometry; mouse prion protein; prion; thioflavin T